Bottom Line:
Moreover, myosin Va tracks the plus end in a Mlp-dependent manner.These results identify a novel +TIP and indicate that vertebrate cells possess a +TIP complex that is similar to the Myo2p-Kar9p-Bim1p complex in yeast.We suggest that the +TIP complex identified in this study may serve to focus the transfer of melanosomes from microtubules to actin at the microtubule plus end.

ABSTRACTIn mouse melanocytes, myosin Va is recruited onto the surface of melanosomes by a receptor complex containing Rab27a that is present in the melanosome membrane and melanophilin (Mlp), which links myosin Va to Rab27a. In this study, we show that Mlp is also a microtubule plus end-tracking protein or +TIP. Moreover, myosin Va tracks the plus end in a Mlp-dependent manner. Data showing that overexpression and short inhibitory RNA knockdown of the +TIP EB1 have opposite effects on Mlp-microtubule interaction, that Mlp interacts directly with EB1, and that deletion from Mlp of a region similar to one in the adenomatous polyposis coli protein involved in EB1 binding blocks Mlp's ability to plus end track argue that Mlp tracks the plus end indirectly [corrected] by hitchhiking on EB1. These results identify a novel +TIP and indicate that vertebrate cells possess a +TIP complex that is similar to the Myo2p-Kar9p-Bim1p complex in yeast. We suggest that the +TIP complex identified in this study may serve to focus the transfer of melanosomes from microtubules to actin at the microtubule plus end.

fig5: Mlp hitchhikes on EB1. (A–F) The overexpression of EB1-GFP recruits Mlp-mRFP onto the length of microtubules (A–C), but this is not seen when Mlp-GFP is overexpressed by itself (D–F; Fig. S2 E shows that Mlp-GFP is not recruited nonspecifically to taxol-stabilized microtubules; available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). (G) Melanocytes make EB1 but not EB3 (CV1 cells do the converse). Blots probed with antibody to EB2/RP1 failed to detect this protein in melanocyte (MC) extracts (not depicted). (H) The reduction in cellular EB1 level after short inhibitory RNA treatment. (I and J) GST-EB1 but not GST binds Mlp-FLAG whether present in SF9 whole cell extracts (I) or as a pure protein (J; bottom, Coomassie blue; top, Westerns with α-FLAG or α-Mlp antibodies). (K) Purified Mlp. (L) Schematic of Mlp and an alignment of COOH-terminal sequences in Mlp and APC (blue region; m, mouse; h, human). Only residues that are shared between Mlp and APC are highlighted (blue, identity; yellow, conservative substitution). Brackets (I–IV) show the positions of four pseudorepeats in APC (Slep et al., 2005). Mlp and APC have no similarity outside of the region aligned in L, and Mlp lacks APC repeat IV, which is critical for APC–EB1 interaction. Asterisks indicate COOH-terminal ends. Numbers below the schematic indicate the residue number in Mlp. Numbers in bold (bottom) indicate the beginning and ending residue numbers.

Mentions:
Double staining of melan-c melanocytes with antibodies to EB1 and Mlp showed that a subset of EB1 comets contain Mlp staining (Fig. 3 D). Of 595 EB1 comets in nine cells, 22.0 ± 7.6% contained Mlp staining. As a control for random overlap between EB1 comets and Mlp-positive melanosomes, cells were double stained for EB1 and Rab27a because Rab27a is required for the targeting of Mlp to melanosomes (Wu et al., 2002a) and because Rab27a itself does not surf (see below). 7.0 ± 3.0% of 494 EB1 comets in 10 cells contained Rab27a staining (P < 0.00002 vs. 22.0 ± 7.6%). We conclude, therefore, that a small subset (∼15%) of endogenous EB1 comets contain endogenous Mlp. We also found that in cells overexpressing EB1-GFP, in which EB1 decorates the entire microtubule lattice, endogenous Mlp can be recruited along the length of the microtubule (Fig. 3 E and see Fig. 5).

fig5: Mlp hitchhikes on EB1. (A–F) The overexpression of EB1-GFP recruits Mlp-mRFP onto the length of microtubules (A–C), but this is not seen when Mlp-GFP is overexpressed by itself (D–F; Fig. S2 E shows that Mlp-GFP is not recruited nonspecifically to taxol-stabilized microtubules; available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). (G) Melanocytes make EB1 but not EB3 (CV1 cells do the converse). Blots probed with antibody to EB2/RP1 failed to detect this protein in melanocyte (MC) extracts (not depicted). (H) The reduction in cellular EB1 level after short inhibitory RNA treatment. (I and J) GST-EB1 but not GST binds Mlp-FLAG whether present in SF9 whole cell extracts (I) or as a pure protein (J; bottom, Coomassie blue; top, Westerns with α-FLAG or α-Mlp antibodies). (K) Purified Mlp. (L) Schematic of Mlp and an alignment of COOH-terminal sequences in Mlp and APC (blue region; m, mouse; h, human). Only residues that are shared between Mlp and APC are highlighted (blue, identity; yellow, conservative substitution). Brackets (I–IV) show the positions of four pseudorepeats in APC (Slep et al., 2005). Mlp and APC have no similarity outside of the region aligned in L, and Mlp lacks APC repeat IV, which is critical for APC–EB1 interaction. Asterisks indicate COOH-terminal ends. Numbers below the schematic indicate the residue number in Mlp. Numbers in bold (bottom) indicate the beginning and ending residue numbers.

Mentions:
Double staining of melan-c melanocytes with antibodies to EB1 and Mlp showed that a subset of EB1 comets contain Mlp staining (Fig. 3 D). Of 595 EB1 comets in nine cells, 22.0 ± 7.6% contained Mlp staining. As a control for random overlap between EB1 comets and Mlp-positive melanosomes, cells were double stained for EB1 and Rab27a because Rab27a is required for the targeting of Mlp to melanosomes (Wu et al., 2002a) and because Rab27a itself does not surf (see below). 7.0 ± 3.0% of 494 EB1 comets in 10 cells contained Rab27a staining (P < 0.00002 vs. 22.0 ± 7.6%). We conclude, therefore, that a small subset (∼15%) of endogenous EB1 comets contain endogenous Mlp. We also found that in cells overexpressing EB1-GFP, in which EB1 decorates the entire microtubule lattice, endogenous Mlp can be recruited along the length of the microtubule (Fig. 3 E and see Fig. 5).

Bottom Line:
Moreover, myosin Va tracks the plus end in a Mlp-dependent manner.These results identify a novel +TIP and indicate that vertebrate cells possess a +TIP complex that is similar to the Myo2p-Kar9p-Bim1p complex in yeast.We suggest that the +TIP complex identified in this study may serve to focus the transfer of melanosomes from microtubules to actin at the microtubule plus end.

ABSTRACTIn mouse melanocytes, myosin Va is recruited onto the surface of melanosomes by a receptor complex containing Rab27a that is present in the melanosome membrane and melanophilin (Mlp), which links myosin Va to Rab27a. In this study, we show that Mlp is also a microtubule plus end-tracking protein or +TIP. Moreover, myosin Va tracks the plus end in a Mlp-dependent manner. Data showing that overexpression and short inhibitory RNA knockdown of the +TIP EB1 have opposite effects on Mlp-microtubule interaction, that Mlp interacts directly with EB1, and that deletion from Mlp of a region similar to one in the adenomatous polyposis coli protein involved in EB1 binding blocks Mlp's ability to plus end track argue that Mlp tracks the plus end indirectly [corrected] by hitchhiking on EB1. These results identify a novel +TIP and indicate that vertebrate cells possess a +TIP complex that is similar to the Myo2p-Kar9p-Bim1p complex in yeast. We suggest that the +TIP complex identified in this study may serve to focus the transfer of melanosomes from microtubules to actin at the microtubule plus end.